(W1230-02-09) In Vitro Assessment of Comparative Immunogenicity for Generic Synthetic Peptide Drug Products

Purpose: To facilitate generic peptide submission and approval, the FDA published guidance on ANDAs for Certain Highly Purified Synthetic Peptide Drug Products That Refer to Listed Drugs of rDNA Origin (May 2021) provides FDA’s recommendations for demonstrating the equivalence of synthetically produced generic peptide drug products that reference a recombinantly produced reference listed drug (RLD). Although the guidance is specific for glucagon, liraglutide, nesiritide, teriparatide, and teduglutide, FDA has other peptide products where the product-specific guidances (PSGs) reference this peptide guidance. A critical aspect in the development and approval of these peptide products is the need for an immunogenicity risk assessment. This risk is informed by the RLD’s clinical experience as well as specific quality aspects of the peptide. If the immunogenicity risk of the RLD peptide is considered to be elevated, additional non-clinical assays (e.g., cell-based or functional binding assays) may be needed for generic applicants to demonstrate that their product will not further elevate the immunogenicity risk. To facilitate the development of these non-clinical immunogenicity assays, we examined commonly used assays for detecting innate immune response mediated impurities (IIRMIs), including cytokine secretion by peripheral blood mononuclear cells (PBMCs). We investigated the formulation (of teriparatide) and storage handling effects on the assay sensitivity and performance.

Methods: PBMCs were isolated from the whole blood of 10 healthy donor volunteers. To assess various blood storage and handling conditions PBMCs were isolated from fresh whole blood on the same day (fresh PBMC), freshly isolated and incubated for 24 hrs prior to use (cultured PBMC),  freshly isolated and cryopreserved before use (cryopreserved PBMC) or isolated from the whole blood stored for either 24 or 48 hours at 4 °C (PBMC from 24 hr stored blood and PBMC from 48 hr stored blood, respectively). Teriparatide drug product (FORTEO) and ten IIRMIs from various sources were tested, each at four concentrations. After PBMCs were prepared for culture based on the above conditions, they were incubated with controls, teriparatide drug product, or IIRMIs for 24 hours. Teriparatide drug product and IIRMI controls were tested at various concentrations. At the end of incubation, the samples were centrifuged for 5 min at 18,000xg, and supernatants were again analyzed for the presence of cytokines using our custom 7-plex from Quansys Biosciences for the detection of IL-1α, IL-6, IL-8, IP-10, MIP-1, MCP-1, and PGE-2.

Results: Using fresh PBMC from healthy donors, we found that the formulation buffer of the teriparatide product can influence cytokine induction. In fact, almost all components of the formulation buffer contributed to the induction of certain cytokines, such as PGE-2 and IL-8 (Figure 1). It is also important to note that for IL-8 (figure 2b), a chemokine usually associated with inflammation, levels increased as much as four-fold due to the presence of mannitol. Another factor that affects the cytokine induction and assay sensitivity is PBMC storage and handling. Notably, we found that the use of fresh PBMCs or PBMC that were within 4 hours of collection when they were cryopreserved resulted in the best cell recovery and viability and eventually resulted in the most sensitive platform to assess IIRMIs. (figure 2). Cultured and cryopreserved PBMCs, and fresh whole blood (prior to processing) may be used as some cell viability is retained. However, validation of handling procedures and the selection of cytokines should be justified to show a consistent response between various donors is obtainable. The use of refrigerated whole blood for 24 or 48 hours are not optimal because of the poor PBMC yield (with less than 10% cell viability remained) and possibly affected cellular responses.

Conclusion: We examined some of the best practices for conducting the PBMC assay, and factors that could potentially affect cell viability and responses to support an IIMI evaluation of generic peptide products. The differences in formulations and cell handling conditions can greatly affect cell survival and cytokine releases. Therefore, it is important for drug developers to investigate and understand how these factors would interfere with the cell-based assays during assay development and validation as this can also demonstrate the formulation interference is acceptable for the chosen cytokine signal. To adequately conduct and validate non-clinical immunogenicity assays can be challenging since there is a lack of standardized approaches and best practices for these non-clinical immunogenicity assays. We encourage the continued collaboration between regulatory, industry and academia in researching and developing standardized methods to these in vitro assays.

Acknowledgments: The views and opinions expressed in this presentation are those of the authors, and should not be construed as the Agency's policy.

Figure 1: All components of formulation buffer of teriparatide induces prostaglandin-E2 (PGE-2) and interleukin 8 (IL-8) release



Figure 2: The effect of storage condition on PBMC cell recovery (A) and cell viability (B)